Analogs of cocaine (2.beta.-carbomethoxy-3.beta.-benzoxytropane) (Compound I) are useful in the study of brain function, in particular the dopamine transporter protein and the serotonin transporter protein. Such studies have yielded useful insights into the metabolism and mechanism of action regarding the psychoactivity and addictive properties of cocaine. In addition, fundamental insights have been learned about the physiology and biochemistry of the dopamine and serotonin transporters in normal and pathogenic conditions. ##STR2##
The ability of cocaine and certain analogs thereof to bind to localized receptors within the brain would make it possible, in principle, to utilize the compounds for in situ imaging of the receptors by PET, SPECT and similar imaging methods. PET imaging is accomplished with the aid of tracer compounds labeled with a positron-emitting isotope (Goodman, M. M. Clinical Positron Emission Tomography, Mosby Yearbook, 1992, K. F. Hubner et al., Chapter 14). For most biological materials, suitable isotopes are few. The carbon isotope, .sup.11 C!, has been used for PET, but its short half-life of 20.5 minutes limits its usefulness to compounds that can be synthesized and purified quickly, and to facilities that are proximate to a cyclotron where the precursor .sup.11 C! starting material is generated. Other isotopes have even shorter half-lives. .sup.13 N! has a half-life of 10 minutes and .sup.15 O! has an even shorter half-life of 2 minutes. The emissions of both are more energetic than those of .sup.11 C!. Nevertheless, PET studies have been carried out with these isotopes (Hubner, K. F., in Clinical Positron Emission Tomography, Mosby Year Book, 1992, K. F. Hubner, et al., Chapter 2). A more useful isotope, .sup.18 F!, has a half-life of 110 minutes. This allows sufficient time for incorporation into a radio-labeled tracer, for purification and for administration into a human or animal subject. In addition, facilities more remote from a cyclotron, up to about a 200 mile radius, can make use of .sup.18 F! labeled compounds. Disadvantages of .sup.18 F! are the relative scarcity of fluorinated analogs that have functional equivalence to naturally-occurring biological materials, and the difficulty of designing methods of synthesis that efficiently utilize the starting material generated in the cyclotron. Such starting material can be either fluoride ion or fluorine gas. In the latter case only one fluorine atom of the bimolecular gas is actually a radionuclide, so the gas is designated .sup.18 F-F. Reactions using .sup.18 F-F as starting material therefore yield products having only one half the radionuclide abundance of reactions utilizing K.sup.18 F as starting material. On the other hand, .sup.18 F! can be prepared in curie quantities as fluoride ion for incorporation into a radiopharmaceutical compound in high specific activity, theoretically 1.7 Ci/nmol using carrier-free nucleophilic substitution reactions. The energy emission of .sup.18 F! is 0.635 MeV, resulting in a relatively short, 2.4 mm average positron range in tissue, permitting high resolution PET images.
SPECT imaging employs isotope tracers that emit high energy photons (.gamma.-emitters). The range of useful isotopes is greater than for PET, but SPECT provides lower three-dimensional resolution. Nevertheless, SPECT is widely used to obtain clinically significant information about analog binding, localization and clearance rates. A useful isotope for SPECT imaging is .sup.123 I!, a .gamma.-emitter with a 13.3 hour half life. Compounds labeled with .sup.123 I! can be shipped up to about 1000 miles from the manufacturing site, or the isotope itself can be transported for on-site synthesis. Eighty-five percent of the isotope's emissions are 159 KeV photons, which is readily measured by SPECT instrumentation currently in use.
Use of .sup.18 F! labeled compounds in PET has been limited to a few analog compounds. Most notably, .sup.18 F!-fluorodeoxyglucose has been widely used in studies of glucose metabolism and localization of glucose uptake associated with brain activity. .sup.18 F!-L-fluorodopa and other dopamine receptor analogs have also been used in mapping dopamine receptor distribution.
Various fluorinated cocaine analogs have been synthesized. Gatley et al., (1994) J. Neurochem. 62:1154-1162 reported synthesis of 4'-.sup.18 F!fluorococaine (Compound II) by nucleophilic aromatic substitution from .sup.18 F!fluoride and 4'-nitrococaine. A yield of 10-15% corrected for decay was reported. The yield was believed to be reduced by hydrolysis of both substrate and product under the basic reaction conditions. PET images acquired with .sup.18 F- or .sup.11 C-labeled 4'-fluorococaine were very similar to those acquired with N-methyl-.sup.11 CH.sub.3 !cocaine. ##STR3##
Compounds of the general class 3.beta.-aryltropane-2.beta.-carboxylates have been shown to be, in some cases, up to 500 times more potent than cocaine at binding the dopamine transporter. In particular, the 4'-fluorophenyl derivative (Compound III) has been widely employed and is commercially available. Trivial names for this compound are WIN 35,428 and CFT. (Clark et al., (1973) J. Med. Chem. 16:1260-1267; Madras et al., (1989) Mol. Pharmacol. 36:518-524). According to Gatley et al (1994) supra, no routes of synthesis were available to high specific activity .sup.18 F-! labeled WIN 35,428 (III) since it lacks a functionality to activate a leaving group at the 4' position to nucleophilic displacement with .sup.18 F!F.sup.-. In addition, the iodo compound (2.beta.-Carbomethoxy-3.beta.-(4-iodophenyl)tropane, also called RTI-55 (Compound IV), has been synthesized (Davies et al., (1994) J. Med. Chem. 37:1262-1268). The latter authors reported synthesis of a series of 2.beta.-methyl ketone and -ethyl ketone compounds, replacing the ester linkage at the 2.beta. position found in cocaine (I) and WIN 35,428 (III). Para fluorophenyl derivatives (such as Compound V) were synthesized by a copper-catalyzed addition reaction using a p-fluorophenyl magnesium bromide precursor. No isotopically labeled compounds of this type were reported. Binding of the compounds to dopamine transporter and serotonin receptor was observed, measured in vitro by competitive inhibition of .sup.125 I!-RTI-55 (IV) binding in homogenates of rat brain striata. ##STR4##
A series of N-substituted derivatives of WIN 35,428 (III) were reported by Milius et al., (1991) J. Med. Chem. 34:1728-1731. Synthesis was based on WIN 35,428 (III) as starting material. The latter was itself synthesized by reaction of p-fluorophenylmagnesium bromide with anhydroecgonine methyl ester. Binding was observed in vitro, measured by competition with .sup.3 !H-cocaine (I) in homogenates of monkey brain caudate putamen. None of the described derivatives was isotope labeled.
Madras et al., (1990) Pharmacology Biochemistry and Behavior 35:949-953 reported comparisons of three N-modified fluorophenyl analogs of WIN 35,428 (III), the N-allyl, N-propyl and the N-unsubstituted compound. All three analogs displaced specifically bound .sup.3 H!cocaine (I) in vitro from caudate-putamen homogenates of monkey brain with affinities greater than that of cocaine. In vivo tests with monkeys confirmed cocaine-like interoceptive effects of the analogs. The analogs were synthesized as described by Milius (1991), supra. No synthesis of isotope-labeled analogs was reported.
Delalande, S. A., German OS 30 01 328, disclosed a series of nortropane derivatives having a variety of aromatic and heterocyclic N-substituents and a variety of aromatic and heterocyclic substituents in amide linkage at the 3 position of the tropane nucleus. Included in the N-substituents are fluoro-substituted aromatics, such as Compound VI, the synthesis of which employed a p-fluorobenzyl chloride intermediate. No synthesis using a fluorine isotope was reported.
Kuhar et al, U.S. Pat. No. 5,380,848, issued Jan. 10, 1995, disclosed nortropane analogs having a variety of N-substituents, various substituents at the 2-position and various aromatic substituents at the 3-position. Certain halogenated substituents were disclosed, both as alkyl halogens (specifically, 2-chloromethyl) and as halogen-substituted aromatic groups at the 3-position. Synthesis of 3.beta.-(3'-Methyl-4'-fluorophenyl)tropane-2.beta.-carboxylic acid methyl ester (Compound VII) was disclosed ##STR5## using 3-methyl-4-fluorophenylmagnesium bromide as the fluorinated starting material, with 29% yield of the stated product. Use of radiocarbon or radioiodine for tracer labeling was described generally, and use of .sup.11 C! as label for PET imaging was discussed. Labeling with .sup.18 F! was not disclosed. No isotopic synthesis was described. The ability of the disclosed compounds to displace .sup.3 H!WIN 35,248 (III) in an in vitro assay using rat brain striata homogenates was compared.
Neumeyer et al, U.S. Pat. No. 5,310,912, issued May 10, 1994, disclosed N-substituted, 2-carboalkoxy-3-aryl nortropanes including N-substituted fluoroalkyl derivatives. 1-bromo-3-fluoropropane was reacted with the corresponding nortropane to generate 2.beta.-carbomethoxy-3.beta.-(-iodophenyl)-8-(3-fluoropropyl)-nortropane (Compound VIII). No binding or activity results were reported with regard to the product. No yield data for the synthesis of the final product or the 1-bromo-3-fluoropropane starting material were reported. Longer 2-substituted alkoxy derivatives, up to 6 carbons, were described. No fluoro-derivatives thereof were disclosed. The patent also disclosed the iodo- analog (IV) of WIN 35,428 (III), synthesized by direct iodination of 2.beta.-carbomethoxy-3.beta.-phenyltropane. The compound, 2.beta.-carbomethoxy-3.beta.-(4'-iodophenyl)-tropane, was abbreviated .beta.-CIT or alternatively RTI-55 (IV). Also, .sup.123 I! label was introduced by converting the iodinated compound to the corresponding tributyl tin derivative followed by reaction with Na.sup.123 I resulting in a labeled compound having about 2000 Ci/mmol. .beta.-CIT (IV) was shown to bind dopamine reuptake sites in tissue homogenates of primate striatum in competition with tritiated WIN 35,428 (III). ##STR6##
Dannals et al., (1992) J. Labelled Compounds and Radiopharmaceuticals 33:147-152 disclosed .sup.11 C! labeled WIN 35,428 (III) and method of synthesis thereof by N-methylation of the corresponding free base using .sup.11 CH.sub.3 I. The synthesis required about 21 minutes with a yield of 20.6% not corrected for decay. The labeled compound was intended for use in PET imaging of the dopamine uptake site. Although the compound also contains a fluorine, no synthesis of an .sup.18 F!-labeled compound was reported.
Dopamine (4-(2-aminoethyl)-1,2-benzenediol) is a neurotransmitter in certain parts of the central nervous system (CNS). Neurons responsive to dopamine are termed dopaminergic. Dopamine is synthesized and released by presynaptic dopaminergic neurons and exerts its effects by binding at post-synaptic receptors. Following impulse transmission, dopamine in the synaptic gap is removed by binding to a dopamine transporter protein. The latter thus acts to regulate the amount of free dopamine in the synaptic gap and to prevent continuous excitation of the post-synaptic neurons.
Abnormalities in CNS dopaminergic neurotransmission have been implicated in movement disorders such as Parkinson's disease. This disorder has been shown to be caused by a significant decrease in the synthesis and transmission of dopamine as a result of degeneration of dopamine neurons in the substantia nigra and corpus striatum regions of the brain. Parkinson's disease has been characterized as a result of progressive loss of neurons in the substantia nigra in which age-related attrition is exacerbated or accelerated by an environmental insult of some sort (Calne et al (1985) Nature 317:246-248). Although drug therapy is of some value, its effectiveness diminishes as the disease progresses. Early diagnosis is therefore of great value in providing effective therapeutic intervention.
The dopamine transporter appears to be the site of action of cocaine, which binds the transporter with high affinity and specificity. The effect of the binding is to inhibit dopamine reuptake and therefore prolong the excitation of post-synaptic dopaminergic neurons. The sensations of euphoria and addiction to cocaine are believed to result from its dopamine transporter binding property. Research to improve understanding of the short and long-term effects on brain biochemistry, physiology and possibly anatomy depends on having suitable tracer compounds for quantitative and qualitative determination of dopamine transporter and for imaging under conditions of acute dosage, withdrawal and therapy.